The present invention relates to the screening for and use of agents which either inhibit or activate protein splicing of inteins (IVPS). Specifically, disclosed herein is the development of 2 specific reporter systems for Gyrase A and DnaB inteins. Agents screened for in accordance with the present invention can be used to control protein splicing for any purpose, in vivo or in vitro, including antimicrobial activity of organisms containing inteins in essential genes. More specifically, the present invention relates to the use of inteins expressed in modified or unmodified native protein splicing precursors or homologous extein systems to screen for mutations that modulate splicing or agents that inhibit or activate splicing. The present invention improves on current reporter systems used to screen for agents that can control splicing by using a modified or unmodified native precursor or precursor homolog in order to take advantage of the more native intein active site formed by natural precursors or inteins in homologous exteins, since agents that are derived from non-native precursors may not have the identical selected activity on native precursors.
Production of mature proteins involves the flow of information from DNA to RNA to protein. Precise excision of DNA and RNA elements which interrupt that information has been previously described (M. Belfort, Annu. Rev. Genet. 24:363 (1990); T. R. Cech, Annu. Rev. Biochem. 59:543 (1990); Hunter et al., Genes Dev. 3:2101 (1989)). More recently, evidence for the precise excision of intervening protein sequences has also been described for the TFPI allele from Saccharomyces cerevisiae (Hirata et al., J. Biol. Chem. 265:6726 (1990); Kane et al., Science 250:651 (1990)) and the recA gene from Mycobacterium tuberculosis (Davis et al., J. Bact. 173:5653 (1991); Davis et al., Cell 71:1 (1992)). Each of these genes contains internal in-frame peptide segments which must be removed to produce the mature protein. Expression of Tfp1 and RecA each results in two peptides: one representing the intervening protein sequence (IVPS) and the other the ligated product of the external protein sequences (EPS). In 1994, the terms xe2x80x9cinteinxe2x80x9d and xe2x80x9cexteinxe2x80x9d were adopted in place of IVPS and EPS, respectively (Perler, et al., Nucleic Acids Res. 22:1125-1127 (1994)). This post-translational processing event has been termed xe2x80x9cprotein splicingxe2x80x9d. Similarly, the xe2x80x9cVentxe2x80x9d(copyright) DNA polymerase gene from the hyperthermophilic archaeon Thermococcus litoralis contains two in-frame IVPS (Perler, et al., PNAS 89:5577 (1992)) and the DNA polymerase gene from the hyperthermophilic archaeon Pyrococcus species GB-D contains one intein (Xu, M., et al., Cell 75, 1371-1377 (1993)).
Over 80 inteins have been identified in bacteria, archaea and eucarya (Perlxc3xa8r, F. B., et al. Nucleic Acids Res 25, 1087-93 (1997), Dalgaard, J. Z., et al., J Comput Biol 4, 193-214 (1997), Pietrokovski, S., Protein Sci. 7, 64-71 (1998) and Perler, F. B. Nucleic Acids Res. 27, 346-47 (1999). Four inteins have been found in Mycobacterium leprae (Davis, E. O., et al., EMBO J. 13, 699-703 (1994) and Smith, D. R., and et al. Genome Res 7, 802-19 (1997)) and three inteins in Mycobacterium tuberculosis (Cole, S. T., et al. . Nature 393, 537-44 (1998)). One intein has been found in Candida tropicalis (Gu, et al., J. Biol. Chem., 268(10):7372-7381 (1993)).
Controllable IVPS (CIVPS) and methods for using the same to modify, produce and purify target proteins has been described (Comb et al., U.S. Pat. No. 5,496,714, issued Mar. 5, 1996; Comb et al., U.S. Pat. No. 5,834,247, issued Nov. 10, 1998). Methods for using inteins to screen for peptides (or derivative, analogic or mimetic thereof) or any agent that can enter cells to block or activate splicing of a natural or experimental reporter protein have also been described (U.S. Pat. No. 5,834,247, supra. at Example 17). These methods specifically describe the screening of peptides using mycobacterial inteins as targets. The preparation of an in vivo peptide library utilizing chicken xcex1-spectrin is also described.
While a general method of screening for antimicrobial agents using the M. tuberculosis RecA intein in a thymidylate synthetase (TS) reporter system has been described (Belfort, U.S. Pat. No. 5,795,731, issued Aug. 18, 1998), this system suffers from several limitations. Importantly, several studies of protein splicing in foreign contexts (such as the Belfort system) indicate that intein splicing is more efficient in the native extein than in foreign exteins (Xu, EMBO J. 13:5517-5522 (1994), Xu, EMBO J. 15:5146-5153 (1996), Telenti, J. Bacteriol. 179:6379-6382 (1997), Chong J. Biol. Chem, 273:10567-10577 (1998), Liu, FEBS Lett. 408:311-314 (1997), Wu, Biochim. Biophys. Acta 1387:422-432 (1998B), Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997), Derbyshire, Proc. Natl. Acad. Sci USA, 94:11466-11471 (1997), Southworth, BioTechniques 27:110-121 (1999), FIG. 7)). For example, the use of foreign exteins yields temperature-dependent splicing of the Psp-GBD Pol, Mxe GyrA and Synechocystis DnaB inteins (Xu, EMBO J. 13:5517-5522 (1994), Xu, EMBO J. 15:5146-5153 (1996), Telenti, J. Bacteriol. 179:6379-6382 (1997), Chong J. Biol. Chem, 273:10567-10577 (1998), Liu, FEBS Lett. 408:311-314 (1997), Wu, Biochim. Biophys. Acta 1387:422-432 (1998B), Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997) and Southworth, BioTechniques, 27:110-121 (1999), and FIG. 7).
While not wishing to be bound by theory, it is believed that such inefficient protein splicing in the foreign extein context occurs because the flanking extein is, in effect, the substrate of the intein. It is, therefore, likely that the intein may exhibit substrate specificity like all other enzymes. The substrate specificity of the intein limits acceptable extein sequences, hence the native extein sequence is the optimal substrate, whereas foreign extein sequences may not be acceptable substrates at all. For example, studies of the Sce VMA and Mxe GyrA inteins indicate that thiol induced N-terminal splice junction cleavage and splicing are, to varying extents, dependent on the single extein residue preceding the intein (Chong, J. Biochem. 273:10567-10577 (1998), Southworth, BioTechniques, 27:110-121 (1999)). Other extein residues have also been shown to affect splicing of the Sce VMA intein (Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997)).
Additionally, exteins may affect the packing at the intein active site, or global folding of the intein and/or precursor, hence the use of a foreign extein may result in improper folding of the intein or precursor and inefficient or no splicing. Moreover, expression of an extein gene that naturally contains an intein in a foreign host, for example E. coli or yeast, may not be efficient (Perler et al. Proc. Natl. Acad. Sci. USA 89:5577-5581 (1992) and Hodges, et al., Nucleic Acid Res. 20:6153-6157 (1992)), whereas expression of the homologous endogenous extein is likely to be more efficient. For example, the Mycobacterium leprae RecA intein fails to splice in E. coli, while it splices in M. leprae (Davis, et al., EMBO J., 13:699-703 (1994)). It is possible that the M. leprae RecA intein would splice in E. coli RecA, although that has yet to be tested. In another example, the Synechocystis sp. strain PCC6803 DnaB gene, containing an intein, was unclonable in E. coli ( Wu, et al., Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998)). The M. leprae GyrA precursor did not splice efficiently in E. coli and was mostly insoluble, while the homologous Mxe GyrA intein spliced efficiently in E. coli GyrA.
Additionally, the use of homologous exteins would eliminate, in many instances, the need to introduce silent mutations in the reporter gene in order to insert the desired intein (see Belfort, supra., Comb, supra, Example 17).
Homologous exteins may have naturally-occuring, conserved restriction enzyme sites that would allow the cloning of the intein into the homologous extein or they may have enough extein similarity to allow insertion of the intein into the homologous extein by recombination. Such systems also eliminate the need for an exogenous reporter gene, since innate extein properties of the native extein may be used for selection. Alternatively, the native extein may be mutated, either de novo or based on mutations in similar extein genes, to make the extein into a selectable marker or reporter gene.
Accordingly, the most desirable intein splicing systems would be those systems which express an intein from one organism in the homologous extein from the foreign host organism used for expression or to express the native precursor gene in a suitable foreign host organism.
Such intein systems would not only be useful in the screening of antimicrobial agents which inhibit intein splicing within a reporter gene (as described in Belfort, supra, Comb, supra.), but as controllable targets to direct expression of an extein product. Agents, for example peptides, that block intein splicing may be used to limit the expression of an extein in such systems. The suppression of such expression may be highly useful in the drug delivery context, where, for example, one wishes to turn on an enzyme which is active in killing cancer cells, or by delivering needed activity, for example insulin.
Similarly, such intein systems may utilize splicing-incompetent inteins to screen for agents with the ability to activate splicing.
In accordance with the present invention, there is provided selection systems and methods for selecting or screening for mutations or agents that control the splicing of inteins which comprise use of the intein""s native host protein (extein) or a homologous extein in any host organism (FIG. 8). Specifically, in one preferred embodiment, there is provided a positive genetic selection system for the screening of agents which inhibit protein splicing which comprises: a host cell which contains (1) a copy of the extein gene (either episomal, chromosomal or synthetic) gene encoding a mutant or naturally drug-resistant form of a target enzyme (which as used herein includes not only enzymes, but proteins, peptides or the like), and (2) a wild-type or mutant form of the extein gene (either episomal, chromosomal, or synthetic) encoding a drug-sensitive form of the target enzyme which is dominantly cytotoxic upon interaction with the drug, wherein the gene encoding the drug-sensitive form of the target enzyme contains an intein, and wherein the inhibition of splicing of the drug-sensitive form of the target enzyme by a given reagent results in the survival of the host cell in the presence of the drug. In one specific embodiment, a positive genetic selection system which utilizes the M. xenopi GyrA intein is provided. This system is also applicable to any GyrA intein inserted at the same or different site in the GyrA extein gene.
In accordance with another preferred embodiment, there is provided a similar positive genetic selection system for the screening of agents which inhibit protein splicing which comprises a host cell which contains (1) a copy of the extein gene (either episomal, chromosomal or synthetic) encoding a wild type form of a target enzyme, and (2) a gene encoding a dominant cytotoxic form of the target enzyme (either episomal, chromosomal or synthetic) wherein the gene encoding the dominantly and cytotoxic form of the target enzyme contains an intein, and wherein the inhibition of splicing of the cytotoxic form of the target enzyme by a given reagent results in the survival of the host cell. In one particularly preferred embodiment, a positive genetic selection system which utilizes the M. tuberculosis DnaB helicase intein is provided. This positive selection system may also employ any DnaB intein inserted at the same or different site in the DnaB extein gene. Similar systems and methods of screening for agents that activate protein splicing are also provided, as are reporter systems utilizing native or homologous exteins and systems utilizing inteins.
Also provided by the present invention are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents. Similar methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing are also provided.
As used herein, xe2x80x9cagentxe2x80x9d includes, but is not limited to, a peptide (free or displayed on a scaffold such as chicken xcex1-spectrin), a peptide derivative, analogic or mimetic, a natural product or a synthetic molecule.